Previously, we had discovered that Acanthamoeba castellanii contains three myosin isoenzymes, a double-headed myosin II and single-headed myosins IA and IB, that are products of different genes. The earlier evidence was based largely on the dissimilarity of one dimensional peptide maps of the products of enzymatic and chemical cleavage of the myosins. These data have now been extended by immunochemical studies which show that there are no common antigenic sites between myosin II and either myosin IA or IB. Myosin IA and IB do show some cross-reactivity but they can be differentiated immunologically. In addition, we have found no evidence for higher molecular weight peptides that cross-react with the antibodies directed against the heavy chains of myosins II, IA and IB. Therefore, it seems increasingly likely that, despite their low molecular weights, these are the functional forms of the enzymes in the cell. The low molecular weight of myosin IA has been definitively confirmed as 150,000 by a combination of sedimentation-equilibrium and sucrose density gradient centrifugation. For the first time, we have obtained preparations of myosin II whose Mg-ATPase activity can be activated by F-actin. Finally, evidence has been obtained for an actin-activatable myosin that may be a single-headed form of myosin II.